COPYRIGHTpsasir.upm.edu.my/id/eprint/20369/1/IB_2011_11_ir.pdfAn alternative technique, which was a pelletization technique, was then carried out to pelletize L. reuteri C 10, L. gallinarium

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UNIVERSITI PUTRA MALAYSIA

COATING AND PELLETIZATION OF PROBIOTIC LACTOBACILLUS STRAINS TO ENHANCE VIABILITY

DURING PROCESSING AND STORAGE

AZIM BIN HARIS

IB 2011 11

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COATING AND PELLETIZATION OF PROBIOTIC LACTOBACILLUS STRAINS TO

ENHANCE VIABILITY DURING PROCESSING AND STORAGE

By

AZIM BIN HARIS

Thesis submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfillment of the Requirement for the Degree of Master of Science

November 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the

requirement for the degree of Master of Science

COATING AND PELLETIZATION OF PROBIOTIC LACTOBACILLUS STRAINS TO

ENHANCE VIABILITY DURING PROCESSING AND STORAGE

By

AZIM BIN HARIS

November 2011

Chairman: Professor Ho Yin Wan, PhD

Institute: Bioscience

The use of probiotics is one of several approaches that have potential to be an alternative to

antibiotics as growth promoters for improving livestock production. The ability of probiotic

microorganisms to remain viable and stable during processing, storage and digestion is very

crucial for their positive contribution to the host. However, even with sophisticated formulations

under excellent storage conditions, the loss rate is about one log unit of cell reduction per year.

In the present study, attempts were made to enhance the viability of three probiotic Lactobacillus

strains, (L. reuteri C 10, L. gallinarium I 26 and L. brevis I 25), by coating the cells with stearic

acid using a fluidized bed granulator, and by pelletization using the extrusion technique.

Preliminary studies were carried out to evaluate the tolerance of L. reuteri C 10 to high

temperatures (58 to 70 ºC) and acidic conditions (pH 4 to 6) because the melting point of stearic

acid is 57.23 ºC and pH is 5.5, and input temperatures of the fluidized bed granulator could be as

high as 70 ºC. Results of the preliminary study demonstrated that freeze-dried L. reuteri C 10

cells incorporated with cryoprotectants (trehalose and sucrose) exhibited higher survivability

compared to freeze-dried L. reuteri C 10 cells without cryoprotectants when exposed for 30 min

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at 64, 66 and 68 ºC. Freeze-dried cells with cryoprotectants were also able to survive for 15 min

at 70 ºC, but not freeze-dried cells without cryoprotectants. In acidic conditions (pH 4, 5 and 6),

freeze-dried L. reuteri C 10 with cryoprotectants also exhibited higher survival rates than those

without cyroprotectants. Thus, cells incorporated with cryoprotectants were used for the coating

process. The use of a fluidized bed granulator to coat freeze-dried L. reuteri C 10 cells resulted

in the formation of a big lump consisting of stearic acid on the upper portion, and excessive

amount of freeze-dried Lactobacillus cells on the lower portion, instead of fine, uniformly-coated

granules. The coating process using a fluidized bed granulator was not successful in this study

due to rapid solidification of stearic acid inside the tube and spray nozzle because the melting

point of stearic acid could not be maintained. Since fluidized bed coating of L. reuteri C 10 was

not successful, the coating process was not carried out on L. gallinarium I 26 and L. brevis I 25,

An alternative technique, which was a pelletization technique, was then carried out to pelletize L.

reuteri C 10, L. gallinarium I 26 and L. brevis I 25 using the extrusion-spheronization technique

and the extrusion-grinding technique. The extrusion-grinding technique was designed and

developed in this study. It was a simple technique, very easy to handle and did not require

expensive sophisticated equipment as in the extrusion-spheronization technique. The results

revealed that the extrusion-grinding technique produced less (P < 0.001) cell loss during

processing than the extrusion-spheronization technique. A loss of 0.5 to 1.3 log units of viable

cells from the granulation to the spheronization or grinding process was observed in both the

extrusion-spheronization and extrusion-grinding techniques used. However, the extrusion-

grinding technique showed significantly (P < 0.001) higher cell viability during the freeze-drying

process compared to the extrusion-spheronization technique (approximately 50 % and 77 % of

cell loss, respectively). Three formulations (A, B and C, with varied compositions of

microcrystalline cellulose, lactose, inulin and skim milk) were used to pelletize Lactobacillus

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strains in the extrusion-spheronization and extrusion-grinding processes. The results showed that,

generally, formulation A was the best (P < 0.001) formulation to pelletize Lactobacillus cells

using both extrusion-spheronization and extrusion-grinding techniques. After 6 months of

storage at 30 ºC, higher cell viabilities were exhibited by all three Lactobacillus strains in pellets

produced by the extrusion-grinding technique than by the extrusion-spheronization technique.

However, at 4 ºC, only L. reuteri C 10 but not L. gallinarium I 26 and L. brevis I 25, exhibited

higher (P < 0.001) cell viability in pellets produced from the extrusion-grinding technique than

from the extrusion-spheronization technique throughout 6 months of storage. Formulations A

and B were better (P < 0.001) than formulation C in maintaining higher cell viability during 6

months of storage at 4 and 30 ºC, regardless of the techniques used. The results for the pellet

densities showed that pellets from the extrusion-grinding technique had higher (P < 0.001)

density compared to those from the extrusion-spheronization technique. Denser pellets (from

extrusion-grinding technique) might provide better protection against adverse environmental

conditions during storage, especially at 30 ºC (room temperature). In conclusion, the use of

fluidized bed granulator to coat L. reuteri C 10 with stearic acid was not successful. The simple

extrusion-grinding technique designed and developed in this study produced better survival rates

of Lactobacillus cells during processing and storage than the extrusion-spheronization technique.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi

keperluan untuk ijazah Master Sains

PENYALUTAN DAN PEMPELETAN PROBIOTIK LACTOBACILLUS UNTUK

MENINGKATKAN KEBOLEHIDUPAN SEMASA PEMPROSESAN DAN

PENYIMPANAN

Oleh

AZIM BIN HARIS

November 2011

Pengerusi: Profesor Ho Yin Wan, PhD

Institut: Biosains

Penggunaan probiotic ialah satu pendekatan yang berpotensi menjadi satu alternatif terhadap

antibiotik sebagai pendorong pertumbuhan untuk meningkatkan pengeluaran ternakan.

Keupayaan probiotik mikroorganisma untuk kekal dan stabil semasa pemprosesan, penyimpanan

dan penghadaman adalah sangat penting untuk kesan positif pada perumah. Bagaimanapun,

walaupun dengan formulasi sempurna di bawah keadaan penyimpanan yang baik, kadar

kematian sel ialah satu log unit setiap tahun. Dalam kajian ini, percubaan untuk meningkatkan

kebolehhidupan tiga strain probiotik Lactobacillus (L. reuteri C 10, L. gallinarium I 26 dan L.

brevis I 25), iaitu dengan penyalutan sel dengan asid stearic menggunakan „fluidized bed

granulator‟, dan pempeletan menggunakan teknik „extrusion‟. Kajian awal telah dijalankan untuk

menilai toleransi L. reuteri C 10 terhadap suhu tinggi (58 hingga 70 ºC) dan di dalam keadaan

berasid (pH 4 hingga 6), kerana suhu takat lebur asid stearik ialah 57.23 ºC dan pH ialah 5.5, dan

suhu input „fluidized bed granulator‟ mungkin meningkat setinggi 70 ºC. Keputusan kajian awal

menunjukkan L. reuteri C 10 sel beku-kering dengan „cryoprotectants‟ (trehalosa dan sukrosa)

mempamerkan kadar kehidupan yang lebih tinggi berbanding dengan L. reuteri C 10 sel beku-

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kering tanpa „cryoprotectants‟ apabila didedahkan selama 30 minit pada suhu 64, 66 dan 68 ºC.

Sel beku-kering dengan „cryoprotectants‟ juga berupaya untuk hidup selama 15 minit pada suhu

70 ºC, tetapi bukan sel beku-kering tanpa „cryoprotectants‟. Dalam keadaan berasid, (pH 4, 5 dan

6), L. reuteri C 10 sel beku-kering dengan „cryoprotectants‟ juga mempamerkan kadar

kebolehidupan yang lebih tinggi daripada tanpa „cryoprotectants‟. Maka, sel dengan

„cryoprotectant‟ akan digunakan untuk proses penyalutan. Penggunaan „fluidized bed granulator‟

untuk menyalut L. reuteri C 10 sel beku-kering menyebabkan pembentukan satu gumpalan besar

yang terdiri daripada asid stearik pada bahagian atas, dan beku-kering Lactobacillus yang

berlebihan pada bahagian bawah, di sebalik membentuk butiran yang seragam dan penyalutan

yang sekata. Proses penyalutan menggunakan „fluidized bed granulator‟ tidak berjaya dalam

kajian ini disebabkan pembekuan asid stearik yang pesat di dalam tiub dan muncung semburan

kerana takat lebur asid stearik tidak dapat dikekalkan. Disebabkan penyalutan terhadap L. reuteri

C 10 tidak berjaya, proses penyalutan tidak dijalankan pada L. gallinarium I 26 dan L. brevis I

25.

Sebagai teknik alternatif, iaitu teknik pempeletan, ia telah dijalankan untuk mempelet L. reuteri

C 10, L. gallinarium I 26 dan L. brevis I 25 menggunakan teknik „extrusion-spheronization‟ dan

„extrusion-grinding‟. Teknik „extrusion-grinding‟ direka dan dicipta dalam kajian ini. Ia

merupakan teknik yang mudah, senang untuk digunkan, dan tidak memerlukan kos yang tinggi

seperti teknik „extrusion-spheronization‟. Keputusan kajian menunjukkan bahawa teknik

„extrusion-grinding‟ menghasilkan kurang (P < 0.001) kematian semasa pemprosesan berbanding

dengan teknik „extrusion-grinding‟. Kematian sebanyak 0.5 sehingga 1.3 log unit sel hidup

daripada penggranulan ke „spheronization‟ atau pengisaran telah dipamerkan dalam kedua-dua

teknik „extrusion-spheronization‟ dan „extrusion-grinding‟ yang digunakan.

Walaubagaimanapun, teknik „extrusion-grinding‟ menunjukan secara signifikan (P < 0.001)

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kebolehhidupan sel yang lebih tinggi semasa pengeringan beku-kering berbanding dengan teknik

„extrusion-spheronization‟ (anggaran 50 % dan 77 % kematian sel). Tiga formulasi (A, B dan C,

dengan pelbagai komposisi „microcrystalline‟ selulosa, laktosa, inulin dan susu krim) telah

diguna untuk pempeletan sel Lactobacillus dalam proses „extrusion-spheronization‟ dan

„extrusion-grinding‟. Keputusan menunjukkan bahawa, secara umumnya, formulasi A adalah

terbaik (P < 0.001) untuk pempeletan sel Lactobacillus menggunakan kedua-dua teknik

„extrusion-spheronization‟ dan „extrusion-grinding‟. Selepas 6 bulan penyimpanan di 30 ºC,

kebolehidupan sel yang lebih tinggi dipamerakan oleh ketiga-tiga palet Lactobacillus yang

dihasilkan oleh teknik „extrusion-grinding‟ berbanding dengan teknik „extrusion-spheronization‟.

Walaubagaimanapun, di 4 ºC, hanya L. reuteri C 10 tetapi bukan L. gallinarium I 26 dan L.

brevis I 25 menunjukkan lebih tinggi (P < 0.001) kebolehidupan sel dalam pelet yang dihasilkan

oleh teknik „extrusion-grinding‟ berbanding dengan teknik „extrusion-spheronization‟ semasa 6

bulan penyimpanan. Formulasi A dan B adalah lebih baik (P < 0.001) daripada formulasi C

dalam mengekalkan kebolehidupan yang lebih tinggi semasa 6 bulan penyimpanan di 4 dan 30

ºC, tanpa mengira teknik yang digunakan. Keputusan ketumpatan palet menunjukkan pelet yang

dihasilkan oleh teknik „extrusion-grinding‟ mempunyai ketumpatan yang lebih tinggi (P < 0.001)

berbanding dengan yang dihasilkan oleh teknik „extrusion-spheronization‟. Pelet berketumpatan

tinggi (dari „extrusion-grinding‟) mungkin memberi perlindungan yang lebih baik terhadap

keadaan persekitaran yang kurang baik semasa penyimpanan, terutama di 30 ºC (suhu bilik).

Konklusinya, penggunaan „fluidized bed granulator‟ untuk menyalut L. reuteri C 10 sel beku-

kering tidak berjaya. Teknik „extrusion-grinding‟ yang direka dan dicipta dalam kajian ini

menghasilkan kebolehhidupan Lactobacillus sel yang lebih tinggi semasa pemprosesan dan

penyimpanan daripada teknik „extrusion-spheronization‟.

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ACKNOWLEDGEMENTS

First and foremost, I wish to express my heartfelt appreciation and deepest gratitude to the

chairman of the supervisory committee, Professor Dr. Ho Yin Wan, for her supervision,

invaluable advice and guidance, strong support and constant encouragement throughout the

duration of my study. My sincere appreciation is extended to the members of the supervisory

committee, Associate Prof. Dr. Sieo Chin Chin, Associate Prof. Dr. Kalavathy Ramasamy and

Mr. Tommy Julianto for their advice, support and assistance during the course of this study.

Thanks are also extended to Associate Prof. Dr. Mohd Ridzwan A. Halim for his kind

assistance, helpful suggestions and guidance in the statistical analysis.

I also would like to express my sincere thanks to Mr. Khairul Kamal Bakri and Madam Haw

Ah Kam, staff of the Laboratory of Vaccines and Immunotherapeutics, Institute of

Bioscience, as well as staff of the Faculty of Pharmacy, Universiti Teknologi Mara, for their

technical support and kind assistance. Special thanks and gratitude are due to Stellar Gen Sdn.

Bhd. for providing the probiotic Lactobacillus strains for this study. My sincere appreciation

also goes to all my lab mates, Tan Hui Yin, Kok Ching Mun, Yong Su Ting, Lau Gee Leng,

Wong Chuan Loo, Lai Pui Wah, Khomala Ramal, Pornpan Saenphoom, Qi Xiaojing, Hung

Xiaodan, Lee Chin Mei as well as all my friends, Noor Hamizah, Aizat Shamin and Mohd

Fauzihan Karim, for their friendship, help and support to ensure the success of this study.

Last but certainly not least, my utmost thanks to my beloved family, Ayah, Ummi and Abang

for their love, sacrifices, continuous support and encouragement in making this struggle turn a

reality. Above all, to Almighty Allah S.W.T., for blessings on me and my family.

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I certify that a Thesis Examination Committee has met on 30/11/2011 to conduct the final

examination of Azim Bin Haris on his thesis entitled “Coating and Pelletization of

Probiotic Lactobacillus Strains to Enhance Viability During Processing and Storage” in

accordance with the Universities and University Colleges Act 1971 and the Constitution

of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee

recommends that the student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Wan Zuhainis binti Saad, PhD

Senior Lecturer

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Shuhaimi bin Mustafa, PhD

Associate Professor

Halal Products Research Institute


(Internal Examiner)

Norhani binti Abdulla, PhD

Professor



(Internal Examiner)

Abdul Jalil Abdul Kader, PhD

Professor

Industrial Relation and Commercialization Centre

Universiti Sains Islam Malaysia

Malaysia

(External Examiner)

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


Date: 25 January 2012

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Ho Yin Wan, PhD

Professor

Institute of Bioscience


(Chairman)

Sieo Chin Chin, PhD

Associate Professor



(Member)

Kalavathy Ramasamy, PhD

Associate Professor

Faculty of Pharmacy

Universiti Teknologi Mara

(Member)

Tommy Julianto

Faculty of Pharmacy

Universiti Teknologi Mara

(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

AZIM BIN HARIS

Date: 30 November 2011

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xv

LIST OF FIGURES xviii

LIST OF ABBREVIATIONS xix

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW

2.1 Use of growth-promoting antibiotics in livestock industry 5

2.2 Defination of probiotics 6

2.3 Beneficial effects of probiotics 7

2.4 Possible modes of action of probiotics 7

2.5 Lactic acid bacteria as probiotic 8

2.6 The genus Lactobacillus 11

2.7 Selection of probiotic strains 14

2.8 Processing of probiotic Lactobacillus 15

2.9 Preservation technologies 16

2.9.1 Chilled frozen cultures 17

2.9.2 Drying techniques 18

2.9.3 Microencapsulation technology 19

2.9.3.1 Spray chilling and cooling 20

2.9.3.2 Spinning disk and centrifugal coextrusion 22

2.9.3.3 Fluidized bed coating 23

2.9.3.4 Extrusion 24

2.9.3.5 Emulsion technique 24

3 OPTIMIZATION OF COATING PROCESS WITH STEARIC ACID

USING A FLUIDIZED BED GRANULATOR ON VIABILITY OF

PROBIOTIC LACTOBACILLUS STRAINS

3.1 Introduction 26

3.2 Materials and Methods 27

3.2.1 Bacterial strain and maintenance 27

3.2.2 Growth study of Lactobacillus strains 27

3.2.3 Morphological study 28

3.2.4 Preparation of inoculums and fermenter conditions 29

3.2.5 Heat and acidic tolerance of Lactobacillus strains 30

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3.2.6 Fluidized bed granulator coating process 31

3.2.7 Statistical Analysis 33

3.3 Results and Discussion 33

3.3.1 Growth of Lactobacillus strains 33

3.3.2 Morphology of Lactobacillus strains using light and

scanning electron microscopy

34

3.3.3 Optimization of the coating process 36

3.3.3.1 Viability of freeze-dried L. reuteri C 10 at high

temperatures

36

3.3.3.2 Viability of freeze-dried L. reuteri C 10 cells

at low pH

39

3.3.3.3 Coating process of L. reuteri C 10 with stearic

acid using a fluidized bed granulator

41

3.4 Conclusions 45

4 EVALUATION OF PELLETIZATION PROCESSES USING THE

EXTRUSION-SPHERONIZATION AND EXTRUSION-GRINDING

TECHNIQUES ON VIABILITY OF PROBIOTIC LACTOBACILLUS

STRAINS

4.1 Introduction 46


4.2.1 Preparation of Lactobacillus strains for pelletization

processes

48

4.2.2 Pelletization using the extrusion-spheronization

technique

48

4.2.3 Pelletization using extrusion-grinding technique 50

4.2.4 Viable cell counts 53

4.2.5 Measurement of moisture contents 53



4.3.1 Optimization of extrusion-spheronization technique 54

4.3.2 Optimization of extrusion-grinding technique 57

4.3.3 Viability of L. reuteri C 10 cells using the extrusion-

spheronization and extrusion-grinding techniques

58

4.3.4 Viability of L. gallinarium I 26 cells using the extrusion-


61

4.3.5 Viability of L. brevis I 25 cells using the extrusion-


65

4.4 Conclusions 68

5 INFLUENCE OF FORMULATIONS AND PELLETIZATION

TECHNIQUES ON VIABILITY OF PROBIOTIC LACTOBACILLUS

STRAINS DURING STORAGE

5.1 Introduction 70


5.2.1 Pellet samples 71

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5.2.2 Storage conditions 71

5.2.3 Enumeration of viable Lactobacillus cells during storage 72

5.2.4 Determination of pellet density 72



5.3.1 Viability of L. reuteri C 10 cells during six months of

storage at 4 ºC

73

5.3.2 Viability of L. gallinarium I 26 cells during six months

of storage at 4 ºC

77

5.3.3 Viability of L. brevis I 25 cells during six months of

storage at 4 ºC

80

5.3.4 Viability of L. reuteri C 10 cells during six months of

storage at 30 ºC

83

5.3.5 Viability of L. gallinarium I 26 cells during six months

of storage at 30ºC

86

5.3.6 Viability of L. brevis I 25 cells during six months of

storage at 30 ºC

88

5.3.7 Pellet densities produced by the extrusion-spheronization

and extrusion-grinding techniques

91

5.4 Conclusions 93

6 GENERAL DISCUSSION AND CONCLUSIONS

6.1 General Discussion 94

6.2 Conclusions 101

REFERENCES 103

APPENDICES 117

BIODATA OF STUDENT 123

LIST OF PUBLICATIONS 124

Documents

COPYRIGHTpsasir.upm.edu.my/id/eprint/20369/1/IB_2011_11_ir.pdfAn alternative technique, which was a pelletization technique, was then carried out to pelletize L. reuteri C 10, L. gallinarium